Beyond depression and anxiety; a systematic review about the role of corticotropin-releasing hormone antagonists in diseases of the pelvic and abdominal organs

Evidence for beneficial effects of corticotropin releasing hormone (CRH) antagonists in abdominal and pelvic organs is emerging in preclinical studies. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement a compilation of preclinical studies using CRH receptor antagonists as a treatment for abdominal and pelvic disease was carried out. The Animal Research: Reporting of In Vivo Experiments (ARRIVE) essential 10 guidelines were used to determine quality of the included studies. A total of 40 studies from the last 15 years studying irritable bowel syndrome, inflammatory bowel disease, endometriosis, enteritis, stress impact on gastrointestinal processes and exogenous CRH administration effects were included. Blockage of the CRH receptor 1 was mainly associated with beneficial effects while that of CRH receptor 2 worsened studied effects. However, time of administration, route of administration and the animal model used, all had an impact on the beneficial outcomes. Frequency of drugs administered indicated that astressin-2B, astressin and antalarmin were among the most utilized antagonists. Of concern, studies included were predominantly carried out in male models only, representing a gender discrepancy in preclinical studies compared to the clinical scenario. The ARRIVE score average was 13 with ~60% of the studies failing to randomize or blind the experimental units. Despite the failure to date of the CRH antagonists in moving across the clinical trials pipeline, there is evidence for their beneficial effects beyond mood disorders. Future pre-clinical studies should be tailored towards effectively predicting the clinical scenario, including reduction of bias and randomization.


Introduction
Corticotropin-releasing hormone (CRH; also known as corticotrophin releasing factor or CRF) is a 41-amino-acid peptide produced by parvocellular neuroendocrine cells of the the colon. IBD pathogenesis is related to genetic, lifestyle, and environmental factors [16]. Worldwide, an increasing incidence of IBD has been observed over the past few decades; therefore, numerous therapeutic advances have emerged (e.g., [17,18]). However, current treatments generate many side effects and do not effectively cure this refractory disease [19]. IBS is a chronic GI disorder affecting the small and large intestines and is characterized by diarrhea, abdominal pain, bloating, flatulence, and constipation [20]. IBS pathogenesis includes GI motility dysfunctions, malabsorption, and gut microbial and enteric nervous system alterations. Even though its etiology remains unclear, evidence suggests that economic, sociologic, and psychological factors are involved [21]. Studies have shown that IBS affects the gut-brain axis, linking conditions like depression and anxiety to the disease, making its treatment more complex [22]. IBS therapy is currently focused on controlling the symptoms but is often ineffective or not tolerated by patients [23].
Preclinical studies have reported that peripheral CRH is implicated in the pathophysiology of abdominal and pelvic diseases such as IBD, IBS, endometriosis, and bladder disorders [24][25][26][27]. Therefore, this literature review summarizes the current pre-clinical evidence for CRH antagonists as a treatment in abdominal and pelvic diseases and discloses some of the barriers preventing these drugs from reaching the commercial arena.

Methods
This is a Systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Articles were extracted from PubMed and Cochrane Library electronic databases. JEPB and JLC extracted the articles during the month of June 2021. Articles included in this study must have been indexed in the afore-mentioned databases from June 1, 2006-June 1, 2021 (15 years).
1. Studies in animals (pre-clinical) or any type of in vivo testing done in any animal model was included in the manuscript.
2. CRH-R1 and/or CRH-R2 antagonists used as a treatment or experimental tool in abdominal and pelvic organs. 3. Written in the English language.
Articles were excluded from this review if they: 1. Focused exclusively on CRH antagonist synthesis and structure.
2. Assessed depression and anxiety without any observation of abdominal or pelvic organs.
3. Studied drug addiction and food craving exclusively, without reported effects on abdominal or pelvic organs. 4. Comprised in vitro studies exclusively. 5. Were qualitative research studies. 6. Were case reports, review articles or commentaries. 7. Assessed CRH agonist treatment.
For informative purposes, clinical studies were summarized in a table format and discussed in relevance to pre-clinical findings.

Quality assessment
The Animal Research: Reporting of In Vivo Experiments guidelines 2.0 (ARRIVE) was used [28,29]. The ARRIVE Essential 10 is a checklist that uses ten items to evaluate study design, sample size, inclusion and exclusion criteria, randomization, blinding, outcomes measures, statistical methods, experimental animals, experimental procedures, and results. A modified methodology for quality assessment by Garcia-Gonzalez et al. [30]. was used to evaluate the articles. A scoring of 1 was given if the article met the required detail or recommendation, and 0 if not. The total number obtained by each article is reported in the corresponding table. The third reviewer (ATR) confirmed the first two reviewers (JEPB and JLC) scoring. The maximum score a study could receive was 21 points since some items in the 'Essential 10' checklist had more than one parameter that needed to be quantified. Since the review search dates predate the original publication of the ARRIVE guidelines, we carried out a sub-analysis of studies published on or before 2010 compared to those published from 2011-2021. Quality of the clinical studies was not assessed as these were only included for informative purposes.

Parameters of interest
Elements collected from articles were first author, year of publication, animal model used (mice or rat), sex, testing compound, country where the study was done, and the main findings of the testing compound used in the study. The first two authors of this study carried out the data extraction. The mean difference between outcomes was not the focus of this review but rather the qualitative effect of the drug in the outcomes of interest. Therefore, the data extractors looked for specific terms in the results indicating: increase, decrease or no change; significant or not significant. In addition, the data extractors also noted when mechanisms of action related to the drugs were reported. These elements are presented in table format.

Data synthesis
Articles were randomly divided into two sets evaluated by JEPB and JLC and systematically screened for inclusion criteria followed by exclusion using a table format in Microsoft Excel. Articles meeting all criteria for screening were then tabulated in a separate spreadsheet and the full extraction process began. The process was verified by the corresponding author to account for missing information. Since the ARRIVE guidelines were used for assessing the articles quality, the percent agreement between the scorers was calculated as well as the Spearman-Brown coefficient for the effectiveness of the "Essential 10" list for ARRIVE as applied to the articles included herein.

Risk of bias
Three elements of the ARRIVE guidelines concern study biases: recommendation number three is related to the adequate reporting of inclusion and exclusion of experimental subjects; recommendation number four requires the reporting of randomization and strategies to minimize confounders; and recommendation number five requires the reporting of the tools used for blinding across the experiments. Failure of reporting across these three recommendations was considered a biased study.

Statistical analyses
Descriptive analyses for the parameters of interest included mean, standard deviations, and percentages, as appropriate. Microsoft Excel was used to organize and summarize data elements. Statistical analyses and graphs were done using GraphPad Prism (v. 8, San Diego, California).

Search results
Using the key word search strategy in PubMed, an initial 2,144 articles were generated from June 1, 2006, to June 1, 2021. Duplicates were eliminated resulting in 950 unique articles. Because of this, the following key words searches became redundant: corticotropin releasing hormone antagonist AND inflammation, verucerfont AND treatment, CP-316,311 AND treatment, NBI-30775 AND treatment, NBI-34041 AND treatment, ONO-2333Ms AND treatment, emicerfont AND treatment, crinecerfont AND treatment, GSK-586529 AND treatment, tildacerfont AND treatment, SSR-125543 AND treatment, NBI-74788 AND treatment, GW-876008 AND treatment, and SPR001 AND treatment. The resulting 950 article abstracts were screened, and 905 did not meet the inclusion criteria established. The 45 articles initially included went through a second screening round and only 40 met the inclusion criteria (Fig 1. Flowchart of search strategy). To study the beneficial effects of CRH-R antagonists different animal models were used. For this review, results will be presented by the type of disease model. This summary strategy was decided a posteriori, based on the obtained results. A total of six articles used CRH administration, 17 used stress models to observe gastrointestinal effects, 13 used administration of chemicals to induce inflammation, and four used surgical procedures to model the disease under study. Tables 1-4 provide details and specific descriptions of the manuscripts included. Compounds used as antagonist were classified by their receptor selectivity (Fig 2. Diagram of compounds by receptor selectivity). As supplementary information, S1-S4 Tables include additional details of each study such as animal body weight (when reported), specific drug concentrations, and the type and duration of stressors in S2 Table. Models CRH administration model. To increase colonic motility intracerebroventricular (ICV) administration of CRH in male rats was done by Ataka et al. which resulted in increased fecal pellet output (FPO) and motor index. In their study, administration of astressin (non-selective CRH antagonist) and NBI-27914 (CRH-R1 selective antagonist) inhibited the afore mentioned effects of CRH but antisauvagine-30 (selective CHR-R2 antagonist) did not [31]. Gourcerol et al. observed that administration of astressin 2B (CRH-R2 antagonist) exacerbated CRH induced FPO and diarrhea while urocortin 2 decreased these effects [32]. In the study by Million et al. pretreatment with NGD 98-2 or NGD 9002 (both CRH-R1 antagonists) reduced the ICV CRH induced FPO, but when CRH was administered by IP injection, only NGD 9002 reduced FPO [33]. On the other hand, Teitelbaum et al. observed that administration of antisauvagine inhibited chronic CRH induced colon permeability [24]. In the study of Tsukamoto, et al. pretreatment of astressin attenuated CRH induced colonic motility in male rats [34]. Finally, Zhao Y. and colleagues using male rats observed that pretreatment with astressin 2B, but not CP-154526 (selective, non-peptide CRH-R1 antagonist) reversed the inhibitory action of CRH on jejunal motor index [35]. See Table 1 for additional details.

PLOS ONE
Stress models. In the study by Yoshimoto, S. male rats were exposed to chronic heterotypic stress (water avoidance, forced swim stress, cold restraint stress and restraint stress) and treated with NBI-27914 and astressin-2B, but only NBI-27914 inhibited the accelerated colonic transit [36]. On the other hand, Barreau et al., observed that male rats with induced stress by neonatal maternal deprivation exhibited increased gut paracellular permeability whereas treatment with α-helical CRH 9-41 and SSR-125543 (selective CRH-R1 antagonist) decreased this effect [37]. In the study of Li B., mice exposed to maternal separation were observed to have increased IL-6, TNFα and inducible nitric oxide synthase (iNOS), which were inhibited by pretreatment with astressin and antalarmin (CRH-R1 antagonist) [26]. However, astressin 2B did not prevent the afore mentioned effects. In addition, antalarmin and astressin improved colonic morphology and decreased myeloperoxidase enzyme; however only antalarmin was able to decrease phosphorylated NF-kB. Van den Wijnguard observed that administration of α-helical CRH 9-41 as a pretreatment, but not as a treatment, prevented visceral hypersensitivity induced by maternal separation and water avoidance in male rats [38]. In addition, animals receiving the antagonist pretreatment had higher occludin levels. In another study, Boucher et al., demonstrated that astressin 2B but not antalarmin decreased VEGF release from bladder significantly reduced FPO. NGD 98-2 and NGD 9002 oral pretreatment prevented the incidence of diarrhea induced by CRF. PO pretreatment of NGD 98-2 effectively reduced centrally or peripherally injected CRF-or acute water avoidance stress-induced colonic motor stimulation and repeated tonic colorectal distensioninduced visceral hypersensitivity, while having no effect on defecation in a non-stressed rat. 13 Teitelbaum A et al.   Canada Increases in IL6, TNF-α and iNOS were inhibited by pretreatment with Astressin and Antalarmin. However, Astressin-2β did not prevent the maternal separation-induced elevation in proinflammatory cytokines. Pretreatment of Antalarmin and Astressin during maternal separation improved colonic morphology, increased crypt length and the number of goblet cells per crypt, and decreased MPO (proinflammatory enzyme) levels. However, pretreatment with Astressin-2β did not rescue the MS-induced colonic injury and demonstrated no effect on immune activation, colonic morphology, and trans-cellular permeability. NF-κB (important protein complex in initiating and regulating inflammation in IBD) ratio of phosphorylated (active) to total NF-κB was higher in the MS group compared to controls, and was rescued by Antalarmin, but not Astressin or Astressin-2β.

Rat model (Males)
Maternal separation α-helical CRF 9-41 and Doxantrazole Belgium CRF antagonist as a pretreatment but not as a treatment prevented the visceral hypersensitivity induced by maternal separation and water avoidance in rats. CRF antagonist as a pretreatment had higher occludin levels. Oral pretreatment of E2508 dose-dependently reduced wrap restraint stress-induced defecation. However, E2508 did not significantly inhibit basal defecation in non-restraint stress-induced rats. SC treatment of E2508 dose-dependently decreased the number of abdominal muscle contractions induced by colonic distention.

15
(Continued ) after acute restraint stress in female mice [39]. Bulbul et al. observed that although astressin pretreatment didn't have any effect on acute restraint stress-induced changes in gastric emptying and intestinal transit, astressin pretreatment recovered the exogenous CRH induced deceleration of intestinal transit [40]. Other studies that used restraint stress observed that astressin-B decreased FPO and blocked the increase in distal colon contractions [41], and Psychological stress increased muscarinic contractions in the bladder, which were partially prevented by Antalarmin daily treatment. Voided volume and micturition frequency per day were significantly lower in the psychological stress rats treated daily with Antalarmin than in rats psychologically stressed without any treatment. Mean voided volume per micturition was significantly higher in psychological stress rats treated daily with Antalarmin than in rats psychologically stressed without any treatment. Japan T-3047928 pretreatment via oral route suppressed increased fecal pellet output induced by conditioning fear stress. However, 1mg of T-3047928 had no effect. Orally administered alosetron significantly reduced fecal pellet number in non-stressed rats. However, there was no impact on normal defecation when T-3047928 was orally given. T-3047928 pretreatment (3 and 10mg) improved both defecation and visceral pain in acute and chronic stress-induced disease models with similar dose range without causing constipation phenotype. USA Astressin B pretreatment prevented the significant increase in fecal pellet output of female CRF-overexpressing mouse stress model compared with their wild type when exposed to a novel environment stressor. Astressin B pretreatment SC prevented the increase in fecal pellet output induced by partial restraint stress and exposure to a novel stressor, compared with control. Astressin B pretreatment had no effect on the number of urine spots (voiding response) induced by a novel environment stressor in female CRF-overexpressing mice.  Korea DSS-induced colitis mortality was decreased in mice injected IP daily with Antalarmin but increased in mice treated with Astressin-2B, compared with the vehicle-treated group. Antalarmin treatment flattened DSS-induced colitis weight loss, whereas Astressin-2B treatment accelerated weight loss.
Histological damage analysis of the colon showed that the Antalarmin group had lower histological scores and the Astressin-2B group higher histological scores compared with the vehicle group. Astressin 2B USA Histological analysis revealed that DSS-induced colitis mice treated daily with Astressin2B had more severe colitis, a decrease in body weight %, and a higher overall damage score (crypt damage, leukocyte infiltration and epithelial regeneration) than vehicle-treated controls. Colonic TNF-α, CXCL-1, and IL-6 mRNA levels, and TNF-α and IL-6 protein expression levels were elevated in mice treated with intracolonic Ast2B, whereas the anti-inflammatory cytokines IL-4, IL-5, and IL-12 remained unchanged. TUNEL positive cells (estimates the number of cells with DNA damage) increased by 55% in Asressint2B-treated mice compared with vehicle controls, indicating increased levels of apoptosis. astressin decreased proximal and distal colonic mast cells [42]. However, intracisternal administration of astressin was observed to abolish restraint stress-induced acceleration of colonic transit while IP administration was not effective [43]. In the study by Tagushi et al., oral pretreatment of E2508 (CRH-R1 antagonist) reduced defecation induced by restraint stress while subcutaneous (SC) administration as a treatment decreased abdominal muscle contraction due to colonic distention [44]. Furthermore, SC pretreatment of astressin-B prevented the increase in FPO induced by partial restraint stress, and similar results were observed in female CRH overexpressing mice with astressin-B pretreatment [45]. The water avoidance model was used by Bradesi et al. to induce psychological stress. Although DMP696 (CRH-R1 antagonist) abolished visceral hyperalgesia it did not alter the pain response caused by colorectal distention [46]. Similarly, Buckely et al. used an open field to induce psychological stress where coadministration of the monoclonal IL-6 receptor with antalarmin synergistically decreased FPO and pain threshold to colorectal distention [47]. On the other hand, three studies used footshock as a model to induce stress where Funatsu et al. observed decreased defecation and freezing behavior by α-helical CRH 9-41, Robbins et al., observed that administration of antisauvagine 30 but not antalarmin decreased bladder hypersensitivity and Seki et al., observed that antalarmin partially prevented muscarinic contractions [48][49][50]. Finally, conditioning fear stress was used by Itomi et al. where oral pretreatment with T-3047928 (CRH-R1 antagonist) suppressed the increase in FPO [51]. See Table 2 for additional details.
Chemical models. In preclinical studies administration of chemical compounds was mainly used to induce IBD or IBS-like symptoms in rodent models. Beginning with IBD models, three studies used dextran sulfate sodium (DSS) to induce colitis in male mice to investigate the effects of CRH antagonists. In the study by Im et al., daily IP injection with antalarmin lowered the histological damage found in the colon, decreased the mortality rate, and flattened the loss of weight caused by DSS-induced colitis [52]. Conversely, daily IP astressin-2B treatment resulted in higher histological damage scores of the colon, a raised mortality rate, and increased weight loss in DSS-induced colitis. Gong et al. demonstrated that daily IP astressin-2B treatment for DSS-induced colitis resulted in increased weight loss, higher disease activity     Ischemia induced by clamping the superior mesenteric artery followed by reperfusion caused hemorrhagic lesions in the small intestine, which were reduced dose-dependently by pretreatment with Astressin (3-30 μg/kg, IV) and Astressin-2B (60 μg/kg, IV), with a significant inhibition of lesions of 71.1% and 72.4% respectively. However, NBI-27914 had no effect. The aggravating effects of Urocortin I in I/R-induced intestinal lesions and in the increase of myeloperoxidase activity (> inflammation) in the small intestinal mucosa were significantly abrogated by the co-treatment with Astressin-2B, but not by NBI-27914. Pretreatment (before ischemia) and posttreatment (after reperfusion) with Astressin-2B significantly suppressed enhancements in bacterial invasion in the intestinal mucosa and the up-regulated expression of iNOS on the small intestine following I/R, while that of NBI27-194 did not. index (DAI) scores, and histological damage scores [53]. In addition, astressin-2B treatment increased expression levels of pro-inflammatory cytokines (TNF-α, CXCL-1, and IL-6), increased cells with DNA damage, and increased intestinal permeability. Lastly, Hoffman et al. showed that daily IC treatment with astressin-2B for DSS-induced colitis resulted in increased weight loss, higher histological damage scores, and increased expression of pro-inflammatory cytokines [54]. Results also demonstrated that anti-inflammatory cytokine levels (IL-4, IL-5, and IL-12) remained unchanged, and the number of cells with DNA damage was increased. Kokkotou, et al. injected Clostridium difficile toxin A in the gut of male mice to induce inflammation. Pretreatment with astressin-2B injected IP prevented C. difficile toxin Ainduced enteritis by decreasing epithelial cell damage, immune cell infiltration, and inflammatory chemokines [55]. Similar results were obtained in CRH-R2 knockout animals. La, et al. used IC administration of acetic acid to induce colitis in male rats [56]. Results demonstrated that IP treatment with astressin reduced post-inflammatory visceral hypersensitivity reducing the abdominal withdrawal reflex (AWR) scores. Saito-Nakaya, et al. worked with an IBD model of colitis by injecting trinitrobenzene sulfonic acid (TNBS) into male rats [57]. SC treatment with CP-154,526 attenuated the increased visceral hypersensitivity caused by TNBS inflammation. In the study by Kubo, et al., male rats were injected with indomethacin to induce small intestinal lesions and bowel hypermotility [58]. Indomethacin-induced hemorrhagic lesions were aggravated by intravenous (IV) pretreatment of astressin-2B and astressin, but no effects were shown when using NBI-27914. Additionally, neither of the antagonists improved bowel hypermotility.
Referring to IBS models, Jia, et al. administered acetic acid to induce IBS-like visceral hypersensitivity in male rats [59]. ICV treatment with α-helical CRH 9-41 and NBI-27914 significantly decreased AWR scores and reduced the electromyographic activity in the external oblique muscle in response to colorectal distension. In an IBS model, CRH antagonists were used by Larauche, et al. to test the impacts of cortagine-induced stress-like alterations of colonic function and visceral hypersensitivity in male rats [60]. IV astressin-B pretreatment blocked the cortagine-induced increase in colonic permeability, and SC CP154,526 pretreatment abolished cortagine-induced diarrhea. Nakade, et al. used glucagon-like peptide (GLP) 1 to induce IBS-like acceleration of colonic transit in male rats [61]. Results demonstrated that ICV astressin pretreatment reduced GLP1-induced acceleration of colonic transit. Liu, et al. used iodoacetamide to induce gastric irritation and, consequently, depression in male rats [62]. Antalarmin administered orally reversed the depression-like behavior in these animals.
Nozu, et al. used lipopolysaccharide (LPS) administration in male rats to observe gut motility inhibitory effects [63]. Pretreatment with IP astressin-2B did not modify the reduced motor index change or the suppressed antral contractions induced by LPS. In 2019, Nozu et al. conducted another study using the same LPS rat model where they observed that astressin-2B also does not modify the increased colonic permeability induced by LPS [64]. Additional details for manuscripts in this section are presented in Table 3.
Surgical models. To understand the healing effect of nociceptin/orphanin FQ (N/FOQ), ethanol-induced gastric mucosa damage in adrenalectomized rats was induced by Grandi and colleagues. In their study, administration of α-helical CRH did not alter the N/FOQ healing effect on gastric lesions. In a study by our laboratory, we have shown that administration of antalarmin for 7 days in female rats with endometriosis surgery decreased the number and weight of vesicles developed [27]. In another study, Takeuchi, et al. induced enteritis in male rats by clamping the superior mesenteric artery which caused hemorrhagic lesions in the small intestine [65]. Here, pretreatment of astressin and astressin-B, but not NBI-27914 inhibited the lesions. Wood, et al. used social stress and partial bladder outlet obstruction to induce urodynamic dysfunction in male rats [25]. Pretreatment with NBI-30775 (CRH-R1 antagonist) administered SC prevented the increase of intermicturition interval, bladder capacity, and micturition volume induced by social stress. Similarly, daily treatment with NBI-30775 prevented the increase of all urodynamic measurements mentioned above caused by partial bladder outlet obstruction surgery. Table 4 provides additional details about the studies included in this section.

Sex representation in the studies
From the six studies that used CRH administration as their experimental model, five used only males and one had both males and females. In the 17 studies that used stress as their experimental model, 11 were done in males, two in females, two had males and females and two did not specify sex. On the 13 studies using 10 different chemical models, only one study used male and female animals while the remaining 12 used males only. From the studies using surgical models, three used males and one study used females. Overall, 77% of studies included were done only in males, 8% in females, 10% in males and females, and 5% were unspecified (Fig 3).

ARRIVE results
The average score for ARRIVE was 13.08 (SD 1.64) points. The group with the lowest score were the ones in the CRF administration group, presented in Table 1, with an average score of 12.3 points. The group of manuscripts with the highest average score were the ones using stress models, presented in Table 2, with an average score of 13.35. When the risk of bias was evaluated using the elements of inclusion and exclusion reporting, randomization, and blinding, 77% of all studies complied with at least one of the elements of inclusion and exclusion criteria reporting. Unfortunately, only 44% of the studies included some type of randomization of the animals and blinding was only reported in 41% of the studies. Of note, most studies blinded the investigators during scoring of tissues or analyses but did not mention any type of blinding related to animal testing.
Recognizing that some studies included in this review pre-date the original publication of the ARRIVE guidelines, a sub analysis of studies published on or before 2010 revealed an average of 10.93 (SD 1.98), while those studies published 2011 onward had a score of 13.26 (SD 0.47). This shows an average increase of only 2.35 points in the quality of the studies in a period of approximately 10 years (2011-2021). Finally, the percent of agreement within scorers was calculated at 82.1%. Since we used the Essential 10 recommendations for the ARRIVE guidelines, we also calculated the Spearman-Brown coefficient at 0.779, which is considered acceptable.

Clinical studies
We found three clinical trials manuscripts that report data on CRH antagonists. All three manuscripts were Phase 2 studies (in the United States) and all three were done in women. Interestingly, while IBS was the target disease, two of them report the brain activity [66,67] while only one reports outcomes on gastrointestinal motility and abdominal pain [68]. Table 5 summarizes the main findings of these studies for informative purposes only.

Discussion
In this review, the goal was to determine what effects corticotropin-releasing hormone antagonists have in chronic diseases of the abdominal and pelvic organs. Overall, the results demonstrated a beneficial response by blocking the CRH-R1, while blocking CRH-R2 was mainly associated with worsening the conditions under study. The timing of drug administration, Table 5. Summary of clinical trials using CRH antagonists against disorders of the abdominal or pelvic area.

Author
Year Clinical phase Testing compound and dose

Main findings
Hubbard CS. et al.

2011
Phase II Women with an average age of 35 years GW876008 20 or 200 mg CRH-1 upregulation in the locus coeruleus complex attributable to chronic stress exposure in IBS patients was reduced by GW876008 administration, bringing levels of activation to that seen in control subjects. GW876008 administration did not affect plasma ACTH or cortisol levels in IBS patients or control subjects, but could attenuate stress-induced hypothalamic activation during expectation of abdominal pain. GW876008 resulted in significant blood-oxygen level-dependent activity reductions within regions of an emotional-arousal circuit during pain expectation in both patients and control subjects.

2013
Phase II Women with an average age of 33 years GW876008 20 or 200 mg In IBS patients, the CRH-R1 antagonist produced greater suppression of blood-oxygen leveldependent activity in a wide range of brain regions during extinction of a conditioned fear response, and this inhibition was associated with a reduction of skin conductance response. During extinction, the effects of GW876008 on brain activity in response to a conditioned stimulus indicated greater suppression of pons and left midbrain in IBS compared to controls. During the acquisition phase of the conditioned fear response, GW876008 significantly suppressed lateralized clusters of activity in the thalamus in both patients and controls, and additionally suppressed midbrain activity in IBS patients. No other meaningful effects were noted on gastric emptying, orocecal transit, and ascending colon emptying. There were no significant treatment effects detected by pexacerfont on the number of stools per day, ease of passage scores, stool consistency or subjective symptoms including bloating, gas, urgency, or abdominal pain. route of administration, and the specific animal model used were seen to have an impact on the beneficial effects observed.
It was noted that the largest number of papers published for CRH receptor antagonists occurred in 2007 (150 papers in PubMed); hence the strategy of this review to cover a 15-year period. However, the rate of publication dropped more than 70% by 2020 indicating decreased research focusing on CRH receptor antagonists, most likely responding to the lack of positive results around mood disorders (as previously reviewed in [15]). Nevertheless, an assessment of CRH-R antagonists as therapeutic agents in peripheral disorders was absent and this review intended to fill that gap. Based on our findings, we propose that CRH-R antagonists still have a strong therapeutic potential in the context of HPA axis dysregulation for disorders outside the CNS.

Interpretation of main findings
Rodents pretreated with CRH-R1 selective antagonists and consequently exposed to CRH administration, acute stress, or chemicals that cause GI irritation or dysfunction presented better disease outcomes than rodents pretreated with CRH-R2 selective antagonists. Among the CRH1 antagonists included in this review, antalarmin and NBI-27914 were the most frequently used, appearing in 15 and 6 of the preclinical studies, respectively. CRH1 antagonists demonstrated beneficial roles in treating stress symptoms, GI diseases, bladder disorders, and endometriosis. In stress models, CRH-R1 antagonists inhibited the increase of pro-inflammatory cytokines, myeloperoxidase levels, inflammatory transcription factors, colonic transit, FPO, and improved colonic morphology and urodynamic dysfunctions [25,26,36,47]. CRH-R1 antagonists decreased colonic lesion scores, mortality rate, weight loss, diarrhea, and depression-like behavior in GI disorders models [52,60,62]. In an endometriosis rat model, a CRH-R1 antagonist reduced the number and weight of endometriotic vesicles [27]. Therefore, blocking CRH-R1 confers a protective effect against inflammatory and stress-related diseases of the abdominal and pelvic organs.
Non-selective CRH antagonist use revealed a pivotal role between beneficial and detrimental effects, basically because they could block both CRH-R1 and CRH-R2 receptors. Most findings found more beneficial effects than harmful ones. However, in many studies, the nonselective CRH antagonists could not prevent stress-induced changes in gastric emptying and intestinal transit, reduced bowel hypermotility and inflammatory transcription factors, and improved GI hemorrhagic lesions induced by chemicals [26,40,58]. Since the primary goal for treating abdominal and pelvic diseases in rodents is to prevent the activation of CRH-R1 and stimulate CRH-R2 activation, CRH-R1 antagonists appear to be the drugs of choice.

Quality of the studies
Outcomes from the quality assessment indicate a discrepancy between the clinical and preclinical scenario. IBS, IBD and gastrointestinal disorders are known to be twice as likely in females compared to males [69]. Yet the vast majority (77%) of the studies included herein were in male rodents only. Only 16% of these studies included female rodents to test the efficacy of CRH-R antagonists in chronic diseases. A lack of inclusion of both sexes in the pre-clinical studies represents a critical barrier in the current understanding of the beneficial effects of CRH-R antagonists for gastrointestinal disorders. Furthermore, a lack of pre-clinical data in females could pose a significant barrier as CRH receptors are present in reproductive tissues in abundance and are known to have a paracrine role in the uterus and ovaries [70]. Therefore, further studies should be inclusive with representation of both sexes, especially when the modeled disease is well known to be more frequent in females.
Close to 60% of the studies did not use any type of blinding or randomization which is worrisome. This could be explained by either a lack of compliance for these two essential elements during the experiments, or by a failure in reporting within the manuscripts. It is impossible to distinguish between these two elements, but they strikingly contrast with the ARRIVE guidelines, which were first introduced in 2010 and modified in 2019. While some of the studies included in this review pre-dated the publication of the guidelines used for evaluation, the field had a minimal increase in the quality of the studies reported over a decade. This suggests that either the investigators are ignoring essential elements for appropriately conducting animal studies, or that the ARRIVE guidelines have not had any significant impact on the field. Additional reviews will be needed to answer this important issue.

Clinical relevance and recommendations
CRH-R1 antagonists have been extensively used in preclinical studies, demonstrating some positive effects for treating not only abdominal and pelvic diseases but also depression and anxiety. However, when studied in clinical trials, the results are not reproducible as seen in animals. Some of the limitations for clinical translation have been carefully reviewed by Spierling and Zorrilla [15] and include issues with pre-clinical screening, the dynamic nature of CRH receptor function not matching the tested patients, acute vs. late presentation of disease, dynamic brain plasticity of CRH receptor activity leading to decreased need for antagonism, among other factors. However, Spierling and Zorrilla emphasized that we still do not fully understand CRH role, especially related to brain disorders. This section explores some of the obstacles and flaws preventing CRH-R1 antagonists from reaching the commercial arena and their possible benefits over current treatments for chronic abdominal and pelvic diseases in humans.
IBS and IBD are chronic refractory diseases whose treatments are based on relieving symptoms to improve patient quality of life. The Mayo Clinic website and the Crohn's and Colitis Foundation of America recommend treating these diseases with medication, a healthy diet, stress and pain management activities, and surgery for severe IBD cases [71]. Although current pharmacological treatments focus on relieving symptoms, they have many side effects and sometimes a limited impact on patient well-being [19,23]. For example, long-term use of corticosteroids for IBD can provoke osteoporosis, cataracts, and adrenal insufficiency [72], whereas antispasmodics for diarrhea-predominant IBS (D-IBS) can cause dizziness, blurred vision, xerostomia, and constipation [73].
Most preclinical studies in this review found that CRH-R1 antagonists can inhibit or diminish gastrointestinal symptoms and reduce stress-induced symptoms caused by CRH administration, perhaps by inhibiting CRH-R1 receptor-mediated inflammation. Further, testing in control rats indicates good safety profiles [37,43], and suggests that CRH-R1 antagonists may improve upon IBD and IBS current treatments by dealing with GI dysfunction and stress levels without the need for polypharmacy. Despite such findings, a path towards the commercial arena has not occurred. As previously pointed out, one of the limitations of the available preclinical studies is the predominant use of male animals when many of these diseases are far more frequent in women. This discordant design flaw might contribute to the lack of progress for these drugs in reaching the commercial arena for treating chronic abdominal and pelvic diseases.
Clinical trials to date include a Phase II trial to evaluate the pharmacodynamic effects of pexacerfont (BMS-562086), a CRH-R1 antagonist, in women with diarrhea predominant IBS [68]. Oral administration of pexacerfont had no significant impact on colonic transit changes, the number of stools per day, stool consistency, or subjective symptoms of bloating and urgency. The reasons for this apparent lack of efficacy on GI dysfunction are still unclear. Pharmacokinetic measurements demonstrated that the drug reached the desired plasma concentration, however there was no certainty that it was present in the GI tract where symptoms were exhibited. Although the oral route is more convenient for patients, it may delay drug effectiveness, reducing bioavailability [74]. Additionally, this trial was of short duration (14 days), perhaps insufficient to observe the effects of pexacerfont in this chronic condition. Encouragingly, 10% of study patients had minimal side effects that resolved without interventions. Pexacerfont also had no impact on subjective measures of emotion, a finding compatible with results from other clinical trials using the selective CRH-R1 antagonist, GW876008 [66,67]. These studies tested the efficacy of this drug in modulating the emotional-arousal circuit in women with IBS focusing on the CNS connections in the emotional-arousal circuit. The results showed that GW876008 could attenuate stress-induced hypothalamic activation during abdominal pain expectation and significantly reduced blood-oxygen-level-dependent activity within regions of an emotional-arousal circuit. Therefore, these drugs may be more effective in stress-sensitive patients showing disruptions in the HPA axis and suffering from comorbid mood disorders such as anxiety and depression.
Future clinical trials should focus on exploring the effectiveness of CRH1 antagonists, specifically antalarmin and NBI-27914, in reducing GI symptoms, improving long-term disease outcomes, and comparing these drugs' advantages over current treatments for IBD and IBS. There are few preclinical studies of CRHR1 antagonists treating endometriosis and bladder disorders; therefore, additional studies must be done before these drugs reach clinical trials for these diseases such as the studies included in this review that demonstrated positive results [25,27,50]. Current evidence in this review supports that there is still hope for CRH-R1 antagonists to reveal their potential as emerging treatments for abdominal and pelvic chronic diseases.

Conclusions
Current pharmacological treatments for IBS and IBD are based on improving disease symptoms but not actually curing these diseases, and unfortunately many treatments are known to cause adverse effects. Most of the preclinical studies identified during this review not only prove the effectiveness of CRH antagonists for the treatment of abdominal and pelvic diseases, but also demonstrate that these drugs have minimal side effects. CRH antagonists can potentially offer novel alternatives for patients with abdominal and pelvic pain. Still, more clinical trials should be undertaken focusing on the beneficial effects of these drugs in the peripheral organs given the abundance of CRH receptors in gastrointestinal and reproductive tissues. Although the failure of these drugs in past trials for mood disorders may have significantly decreased enthusiasm to continue pursuing a clinical application, re-focusing their expected outcomes on peripheral organs might offer an exciting avenue for future therapeutics.

Registration and protocol
This review was not registered. The protocol used for this review is explicitly outlined in the methods. The techniques for eliminating duplicity in searches and other type of raw data used for this review are available to interested individuals at the Zenodo repository at the following address: https://doi.org/10.5281/zenodo.6310254.